Macrophages (MF) play a central role in atherogenesis through accumulation of lipids and production of inflammatory cytokines. Plasma phospholipid transfer protein (PLTP) is associated with MF in lesions and its'expression is up-regulated by lipid loading. We reported that PLTP produced within lesions is atheroprotective. The long-term goal of this project is to identify mechanisms whereby cell associated PLTP is anti-inflammatory and atheroprotective. The first aim will test the hypothesis that MF PLTP disrupts TLR2/1 plasma membrane dimerization and signaling, but not preformed TLR3 dimer mediated signaling in endosomes. We will assess the anti-inflammatory effects of MF PLTP on TLR2/1 and TLR3 signaling in PLTP- /-LDLr-/- mice irradiated and reconstituted with bone-marrow (BM) from PLTP+/+ or PLTP-/- mice. Cytokine production and atherosclerosis will be studied. The second aim will test the hypothesis that PLTP alters cholesterol dependent membrane domains (lipid rafts). We will determine if wild-type (WT) or mutant PLTP (which binds ABCA1 but lacks lipid transfer activity) disrupts lipid rafts and/or inhibits TLR2/1 dimerization in vivo and in vitro. The third aim will test the idea that reduced hepatic production of PLTP to reduce plasma levels of PLTP will favor the beneficial effects of cell associated PLTP. We will assess the anti-inflammatory effects of PLTP on TLR2 signaling in LDLr-/- mice with suppressed, but not deleted, hepatic PLTP. This will be accomplished with in vivo administration of mouse PLTP antisense oligonucleotides (ASO). Novel, targeted mouse models for study of chronic inflammation are used to establish the physiological significance of our hypotheses and will be complemented by in vitro studies to identify the mechanisms involved in these physiological events. Laser scanning confocal immunofluorescent microscopy is used to characterize early atherosclerosis progression. Fluorescent resonance energy transfer (FRET) is used to characterize changes in lipid rafts in response to PLTP. Finally, the proposed studies will use antisense oligonucleotides (ASO) to favor the beneficial effects of anti-inflammatory cellular PLTP while reducing the undesirable affects of circulating plasma PLTP. It is imperative that we now know whether cell-directed or plasma-directed PLTP atheroprotective therapeutics should be pursued for human treatment of cardiovascular risk factors. PUBLIC HEALTH RELEVANCE: The proposed studies will significantly alter the direction of PLTP research by providing sound evidence of its role in the regulation of intracellular inflammatory pathways. The results of our studies will lead to a new and improved understanding of MF physiology and inflammation thus providing the bases for novel treatment options for atherosclerosis, diabetes and other chronic diseases that have a strong inflammatory component.